DESCRIPTION (Applicant's Abstract): This is a request for an Independent Scientist Award. The long term objective of this research is the understanding of the biological significance of the neurotransmitter dopamine (DA) in several structures of the central nervous system with respect to its role in the action of drugs of abuse. Two areas of investigation are planned. One area is the examination of the structural features of substrates and transporter affecting kinetics of the human dopamine and norepinephrine transporters, hDAT and hNET, respectively. A great deal of work has gone into the study of inhibitors of hDAT and hNET, but features of substrates have been less studied. In particular, hypotheses concerning the interaction of the amine group and the catechol groups with specific amino acids in the transporter will be tested. A recently developed method, rotating disk electrode voltammetry, will be used to observe the time course of uptake and induced efflux. The results of these studies will be put in the context of kinetic studies on a range of substrates. A second area, related to the above studies, is the localization of the binding site of substrates for hDAT and hNET. Previous work by others has identified regions of inhibitor binding using photoaffinity labels. Part of the proposed work is a collaboration to use recent progress in mass spectrometry to identify the specific amino acids covalently linked to the photoaffinity agents. This work will then be extended by using a newly synthesized photoaffinity substrate to localize substrate binding.